US20250281571A1 - Therapeutic drug for myotonic dystrophy type 1 - Google Patents

Therapeutic drug for myotonic dystrophy type 1

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Publication number
US20250281571A1
US20250281571A1 US18/557,146 US202218557146A US2025281571A1 US 20250281571 A1 US20250281571 A1 US 20250281571A1 US 202218557146 A US202218557146 A US 202218557146A US 2025281571 A1 US2025281571 A1 US 2025281571A1
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Prior art keywords
ppr
vector
asparagine
amino acids
cug
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Masayuki Nakamori
Yusuke Yagi
Takayoshi Imai
Erika Okii
Takayuki Tamai
Risa Ninomiya
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Editforce Inc
University of Osaka NUC
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Editforce Inc
Osaka University NUC
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Assigned to OSAKA UNIVERSITY reassignment OSAKA UNIVERSITY ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NAKAMORI, Masayuki
Assigned to EDITFORCE, INC. reassignment EDITFORCE, INC. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: NINOMIYA, Risa, IMAI, TAKAYOSHI, OKII, Erika, YAGI, YUSUKE, TAMAI, TAKAYUKI
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/168—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/0008—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition
    • A61K48/0025—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid
    • A61K48/0033—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'non-active' part of the composition delivered, e.g. wherein such 'non-active' part is not delivered simultaneously with the 'active' part of the composition wherein the non-active part clearly interacts with the delivered nucleic acid the non-active part being non-polymeric
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P21/00—Drugs for disorders of the muscular or neuromuscular system
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/01—Fusion polypeptide containing a localisation/targetting motif
    • C07K2319/09—Fusion polypeptide containing a localisation/targetting motif containing a nuclear localisation signal
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K2319/00—Fusion polypeptide
    • C07K2319/80—Fusion polypeptide containing a DNA binding domain, e.g. Lacl or Tet-repressor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

  • the present invention relates to a pharmaceutical composition or a method for treating myotonic dystrophy type 1.
  • DM1 Myotonic Dystrophy type 1
  • DM1 Myotonic Dystrophy type 1
  • DM1 is the most common muscle disease in adults.
  • DM1 is an autosome-dominant multi-organ disorder that influences the skeletal muscle, the smooth muscle, as well as the eyeball, the heart, the endocrine system, and the central nervous system, and its symptoms are diverse, such as muscular atrophy, muscular weakness, myotonia, cataract, insulin resistance, hypogonadism, cardiac conduction disorder, anterior baldness, and intellectual impairment.
  • the cause of DM1 is abnormal elongation of the CTG repeat sequence in the 3′ UTR (untranslated region on 3′ side) of the myotonin protein kinase (DMPK) gene.
  • DMPK myotonin protein kinase
  • Splicing abnormality is evoked when a particular splicing control factor binds to a transcription product (mRNA) having an abnormally elongated CUG repeat sequence, resulting in deficiency of splicing control factors required for normal splicing.
  • mRNA transcription product
  • Non-Patent Literatures 1 and 2 Although development of therapeutic drugs for DM1 is currently brought forward with various approaches such as low molecular weight compounds and nucleic acid medicines (such as Non-Patent Literatures 1 and 2), there is no fundamental therapeutic drug at this stage, and treatment of DM1 is confined mainly to palliative therapy.
  • the object of the present invention is to provide a pharmaceutical composition or a method that is effective for treating myotonic dystrophy type 1 (DM1).
  • DM1 myotonic dystrophy type 1
  • Pentatricopeptide repeat (PPR) protein is a protein that comprises a repeat of PPR motif that is about 35 amino acids long, and it is known that one PPR motif specifically or selectively binds to one base.
  • PPR motif specifically or selectively binds to one base.
  • development of RNA editing/modifying technology that utilizes the PPR protein is under way (such as WO 2013/058404).
  • DM1 may be treated by utilizing a modified PPR protein.
  • the present invention is based on the above knowledge, and may have the following characteristics.
  • [6] The pharmaceutical composition according to any of [1] to [5], characterized in that said nucleic acid encoding a protein that specifically binds to the CUG repeat sequence is integrated into an expression vector.
  • [7] The pharmaceutical composition according to any of [1] to [5], characterized in that said nucleic acid encoding a protein that specifically binds to the CUG repeat sequence is integrated into a viral vector.
  • [8] The pharmaceutical composition according to [7], characterized in that said viral vector is a viral vector with tropism towards muscle tissue.
  • AAV vector is an AAV1 vector, an AAV2 vector, an AAV6 vector, an AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV10 vector, an AAV11 vector, or an AAV12 vector.
  • [16] A cell comprising the viral expression vector according to [15].
  • [17] A viral vector that is produced from the cell according to [16].
  • [19] A method for treating myotonic dystrophy type 1, comprising a step of applying to a subject a therapeutically effective amount of the pharmaceutical composition according to [1].
  • myotonic dystrophy type 1 (DM1) may be treated or alleviated by the present invention.
  • FIG. 1 shows that the PPR protein of the present invention specifically bound to the CUG repeat sequence.
  • FIG. 2 shows that the PPR protein of the present invention suppressed the formation of RNA-foci in DM1 model cells.
  • FIG. 3 shows that the PPR protein of the present invention improved splicing abnormality in DM1 model cells.
  • FIG. 4 shows that the PPR protein of the present invention improved muscle differentiation efficiency in DM1 model animals.
  • FIG. 5 shows that the PPR protein of the present invention suppressed the formation of RNA-foci in DM1 model animals.
  • FIG. 6 shows that the PPR protein of the present invention improved splicing abnormality in DM1 model animals.
  • FIG. 9 shows that AAV9-CUG-PPR 1 dose-dependently suppressed the formation of RNA-foci in DM1 model animals.
  • “Ctrl” indicates the PBS administration group
  • “LD” indicates the AAV9-CUG-PPR 1 low dose administration group
  • “MD” indicates the AAV9-CUG-PPR 1 medium dose administration group
  • “HD” indicates the AAV9-CUG-PPR 1 high dose administration group.
  • the vertical axis shows the RNA-foci positive cell rate.
  • FIG. 10 shows that AAV9-CUG-PPR 1 dose-dependently improved splicing abnormality in DM1 model animals.
  • the left figure shows the results of the abnormal splicing detection system of Clcn1, the right figure of Atp2a1.
  • Ctrl indicates the PBS administration group
  • LD indicates the AAV9-CUG-PPR 1 low dose administration group
  • MD indicates the AAV9-CUG-PPR 1 medium dose administration group
  • “HD” indicates the AAV9-CUG-PPR 1 high dose administration group.
  • the vertical axis shows the respective content (%) of the normal splicing isoform.
  • FIG. 11 shows that AAV9-CUG-PPR 1 dose-dependently improved myotonia in DM1 model animals.
  • the vertical axis shows the myotonia score, and the frequency of myotonic discharge decreases in the order of Scores 3, 2, 1, and 0. The frequency of the number of times of myotonic discharge and the severity are correlated.
  • “Ctrl” indicates the PBS administration group
  • “LD” indicates the AAV9-CUG-PPR 1 low dose administration group
  • MD indicates the AAV9-CUG-PPR 1 medium dose administration group
  • “HD” indicates the AAV9-CUG-PPR 1 high dose administration group.
  • FIG. 12 shows the relationship between the administration dosage of AAV9-CUG-PPR 1 and the amount of PPRmRNA expression.
  • GAPDH is used as the internal standard.
  • Each of the individual points show the amount of PPR mRNA expression in the femoral muscle of each mouse individual.
  • the vertical axis is shown in Log representation.
  • “Ctrl” indicates the PBS administration group
  • “LD” indicates the AAV9-CUG-PPR 1 low dose administration group
  • MD indicates the AAV9-CUG-PPR 1 medium dose administration group
  • “HD” indicates the AAV9-CUG-PPR 1 high dose administration group.
  • FIG. 13 shows that AAV9-CUG-PPR 1 time-dependently suppressed the formation of RNA-foci in DM1 model animals.
  • “Ctrl” indicates the PBS administration group.
  • “2 w” “4 w” “8 w,” and “16 w” each shows the test duration after administration of AAV9-CUG-PPR 1.
  • the vertical axis shows the RNA-foci positive cell rate.
  • FIG. 15 shows that AAV9-CUG-PPR 1 time-dependently improved myotonia in DM1 model animals.
  • the vertical axis shows the myotonia score, and the frequency of myotonic discharge decreases in the order of Scores 3, 2, 1, and 0. The frequency of the number of times of myotonic discharge and the severity are correlated.
  • “Ctrl” indicates the PBS administration group.
  • “2 w” “4 w” “8 w,” and “16 w” each shows the test duration after administration of AAV9-CUG-PPR 1.
  • FIG. 16 shows that splicing abnormality is comprehensively normalized after administration of AAV9-CUG-PPR 1.
  • FIG. 16 a is a figure that shows how splicing events that had a change of 0.2 or more PSI (Percent spliced-in) between WT and PBS-administered HSA-LR mice (left) changed in the PPR-administered HSA-LR mice (right).
  • FIG. 16 b is a figure that shows change in regard to DM1-related splicing events identified in Tanner et al. (2021).
  • FIG. 16 c shows the result of gene ontology analysis on the genes of each point in Figure a.
  • the P-value indicates the P value of each gene ontology term.
  • the number of genes with exon skipping improvement rate are represented in the 3 stages of greater than 50%, 20-50%, and 20% or less for PPR-administered HSA-LR mice.
  • FIG. 17 shows the amount of PPR mRNA expression in each organ of AAV9-CUG-PPR 1-administered mice.
  • the vertical axis shows the relative expression amount when the expression amount of 1 ⁇ 10 13 vg/kg-administered mice was set as 1 in Log representation.
  • GAPDH mRNA expression amount is the internal standard.
  • the horizontal axis shows the AAV9-CUG-PPR 1 dosage (vg/kg of mouse body weight). The values of each mouse individual are plotted with circles, and the median is connected with a line.
  • FIG. 18 shows the persistency of mRNA expression in each organ of AAV9-CUG-PPR 1-administered mice.
  • the vertical axis presents the relative expression amount when the expression amount of one AAV administration Week 2 (Day 14) mouse individual was set as 1.
  • GAPDH mRNA expression amount is the internal standard.
  • “Ctrl” indicates the PBS group, and “14” “28” “56” “112,” and “183” shows the number of days after administration.
  • the values of each mouse individual are plotted with circles, and the average value is connected with a line.
  • PPR motif in the present disclosure, unless particularly described, refers to a polypeptide, when analyzing the amino acid sequence with a protein domain search program on the web, composed of 30-38 amino acids having an amino acid sequence having an E value less than or equal to a predetermined value (desirably E-03) obtained as PF01535 in Pfam and PS51375 in Prosite. While the position numbers of the amino acids configuring the PPR motif as defined in the present disclosure are almost equivalent to PF01535, they correspond to numbers which are two subtracted from the amino acid positions of PS51375 (e.g.
  • position 1 of the present invention ->position 3 of PS51375), provided that when referring to the amino acid at “ii” ( ⁇ 2) position, it is the second amino acid from the tail end (the C-terminal side) of the amino acids configuring the PPR motif, or two amino acids to the N-terminal side of the first amino acid of the next PPR motif, i.e. the amino acid at position ⁇ 2.
  • the amino acid that is two amino acids before the first amino acid of the next helix structure is set at “ii.”
  • a typical PPR motif is composed of 35 amino acids, but its length is variable at 30-38 amino acids.
  • the basic skeleton of the PPR motif is known to be represented by Formula 1.
  • PPR protein in the present disclosure, unless particularly described, refers to a PPR protein having one or more, preferably two or more of the aforementioned PPR motifs.
  • Protein in the present disclosure, unless particularly described, refers to a general substance consisting of a polypeptide (a chain having multiple amino acids bound by peptide bonds), and also includes those consisting of polypeptides of relatively low molecular weight.
  • Amino acid in the present invention may refer to an ordinary amino acid molecule, as well as to an amino acid residue that configures the peptide chain. To which it is referring to will be clear to those skilled in the art from the context.
  • “Selective” or “specific” in the present disclosure in regard to the binding ability of the PPR motif to an RNA base refers to the fact that the binding activity against any one base of the RNA bases is higher than the binding activity against other bases. This selectivity or specificity can be confirmed by those skilled in the art by planning experiments based on well-known methods, as well as be obtained from calculation by those skilled in the art.
  • RNA base in the present disclosure refers to the ribonucleotide base configuring the RNA, and specifically refers to any of adenine (A), guanine (G), cytosine (C), or uracil (U). Note that although the PPR protein may have selectivity against a base in the RNA, it will not bind to the nucleic acid monomer.
  • PPR proteins are abundantly present in plants, and 500 proteins, about 5000 motifs are found in Arabidopsis thaliana . PPR motifs and PPR proteins of diverse amino acid sequences are also present in many land plants such as rice, popular, and selaginella. In the present invention, naturally occurring PPR motifs and PPR proteins may be employed, or PPR motifs and PPR proteins designed based on the method disclosed in for example WO 2013/058404 may be utilized. Specifically, desired PPR motifs and PPR proteins can be designed based on the following information disclosed in WO 2013/058404.
  • the combination of three amino acids at 1, 4, and “ii” ( ⁇ 2) positions (A 1 , A 4 , L ii ), or the combination of two amino acids at 4 and “ii” ( ⁇ 2) positions (A 4 and L ii ) of the PPR motif is important for selective binding with the RNA base, and which RNA base it binds to can be determined by these combinations.
  • the present invention can utilize the knowledge related to the combination of three amino acids at A 1 , A 4 , and L ii and/or the combination of two amino acids at A 4 and L ii as disclosed in WO 2013/058404.
  • the PPR motif thereof has selective RNA base binding ability that is binding strongly to G, but not binding to A, U, and C.
  • the PPR motif thereof has selective RNA base binding ability that is binding strongly to A, but not binding to G, U, and C.
  • the PPR motif thereof has selective RNA base binding ability that is binding strongly to U, in the next place binding to A, but not binding to G and C.
  • the PPR motif thereof has selective RNA base binding ability that is binding strongly to A, but not binding to G, U, and C.
  • the PPR motif thereof has selective RNA base binding ability that is binding strongly to U, in the next place binding to C, but not binding to A and G.
  • the PPR motif thereof has selective RNA base binding ability that is binding strongly to U, but not binding to A, G, and C.
  • the PPR motif thereof has selective RNA base binding ability that is binding strongly to G, but not binding to A, U, and C.
  • the above rules mean that the possibility that the PPR motif binds to the target base may be increased with statistical significance by the amino acid combination at (A 1 , A 4 , L ii ) or (A 4 and L ii ) of the PPR motif, and it does not mean that a PPR motif (or PPR protein) that binds to the target base (or base sequence) with 100% probability may be manufactured by the above rules.
  • those skilled in the art will be able to obtain a PPR motif (or PPR protein) that binds to the target base (or base sequence) with high probability by manufacturing about several to several tens of types of candidate PPR proteins based on the above rules.
  • the selection of the desired PPR protein through manufacture and confirmation of candidate PPR proteins may be performed within the scope of ordinary trial and error by those skilled in the art, and shall not be excessive burden to those skilled in the art.
  • One PPR motif may recognize a particular base of RNA. Further, based on the present invention, by appropriately selecting the amino acids at particular positions, selective PPR motifs can be selected or designed for each of A, U, G, and C. Furthermore, a protein comprising an appropriate series of such PPR motifs may specifically or selectively recognize the corresponding base sequence. Further, a PPR motif that is capable of selectively binding to a desired RNA base, and a protein having multiple PPR motifs capable of sequence-specifically binding to a desired RNA can be designed with the aforementioned knowledge. Upon designing, the sequence information of the native form PPR motif may be referred for portions other than the amino acids at the important positions in the PPR motif.
  • designing may be performed by employing the native form PPR motif in general and substituting only the said amino acids at the important positions.
  • the number of repeats of the PPR motif can be set appropriately according to the target sequence, for example it can be two or more, and can be 2-30.
  • the present invention relates to a PPR protein that specifically binds to an mRNA comprising abnormally elongated CUG repeat sequence that is the cause of DM1.
  • the PPR protein according to the present invention can be produced by linking multiple sets of [a PPR motif that binds to C, a PPR motif that binds to U, a PPR motif that binds to G] based on the aforementioned theory.
  • the first PPR motif of the PPR protein according to the present invention does not necessarily need to be a PPR motif that binds to C, and the first PPR motif may be a PPR motif that binds to U or a PPR motif that binds to G as long as the PPR protein specifically binds to the CUG repeat sequence as a whole.
  • the number of PPR motifs contained in the PPR protein does not need to be a multiple of 3.
  • Non-limiting examples of the C-binding PPR motif that may be employed in the present invention can include PPR motifs having the following sequences.
  • a PPR motif having a sequence homology (or sequence identity) of 80% or more (preferably, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) with the above sequence, which has the ability to bind to C may be employed.
  • a PPR motif having substitutions, additions, and/or deletions of 7 bases or less (i.e., 1, 2, 3, 4, 5, 6, or 7 bases) to the above sequence, which has the ability to bind to C may be employed.
  • the amino acid substitution may be for example a conservative substitution of amino acids known in the art.
  • Non-limiting examples of the U-binding PPR motif that may be employed in the present invention can include PPR motifs having the following sequences.
  • a PPR motif having a sequence homology (or sequence identity) of 80% or more (preferably, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) with the above sequence, which has the ability to bind to U may be employed.
  • a PPR motif having substitutions, additions, and/or deletions of 7 bases or less (i.e., 1, 2, 3, 4, 5, 6, or 7 bases) to the above sequence, which has the ability to bind to U may be employed.
  • the amino acid substitution may be for example a conservative substitution of amino acids known in the art.
  • Non-limiting examples of the G-binding PPR motif that may be employed in the present invention can include PPR motifs having the following sequences.
  • a PPR motif having a sequence homology (or sequence identity) of 80% or more (preferably, 85% or more, 90% or more, 95% or more, 96% or more, 97% or more, 98% or more, 99% or more) with the above sequence, which has the ability to bind to G may be employed.
  • a PPR motif having substitutions, additions, and/or deletions of 7 bases or less (i.e., 1, 2, 3, 4, 5, 6, or 7 bases) to the above sequence, which has the ability to bind to G may be employed.
  • the amino acid substitution may be for example a conservative substitution of amino acids known in the art.
  • search/analysis related to the identity of the base sequence or the amino acid sequence can be performed by algorithms or programs well-known to those skilled in the art (such as BLASTN, BLASTP, BLASTX, ClustalW).
  • the parameters when employing the programs can be appropriately set by those skilled in the art, and default parameters of each program may also be employed.
  • the specific methods of these analysis methods are also well-known to those skilled in the art.
  • amino acids with similar property refer to for example an amino acid with similar physical properties such as hydropathy, charge, pKa, and solubility, and for example refer to the following.
  • the effects of the present invention are exerted when the PPR protein of the present invention binds to an mRNA comprising an abnormally elongated CUG repeat sequence within the subject cell.
  • the delivery means for the PPR protein into the subject cell is not limited, and a nucleic acid encoding the PPR protein of the present invention may be delivered into the subject cell and allow the expression of the PPR protein of the present invention within the subject cell, or the PPR protein of the present invention per se may be delivered into the subject cell.
  • the delivery means for the protein or nucleic acid is not limited, and various means known in the art can be employed.
  • Non-limiting examples of the delivery means for the nucleic acid or protein into the subject cell can include liposomes, lipid nanoparticles (LNP), high molecular weight micelles, emulsions, high molecular weight microcapsules, antibody-nucleic acid complexes, antibody-drug complexes, viruses, and the like.
  • LNP lipid nanoparticles
  • emulsions high molecular weight microcapsules
  • antibody-nucleic acid complexes antibody-drug complexes, viruses, and the like.
  • Non-limiting examples of viral vectors that may be employed in the present invention can include adeno-associated virus (AAV) vectors, adenovirus vectors, retrovirus vectors, lentivirus vectors, or herpes simplex virus vectors, and it can be appropriately selected by those skilled in the art depending on the tropism towards cells or tissues, the necessity of integration of nucleic acid into the host genome, and the like.
  • AAV adeno-associated virus
  • AAV vectors can be particularly favorably employed in the present invention because they have the ability to infect both dividing cells and resting cells, and can transfer genetic material to extremely diverse cell types. Twelve types of AAV serum types (AAV1-AAV12) have been reported to date, and all of the known serum type can infect various types of tissue cells.
  • the serum type of the AAV vectors that may be employed in the present invention is not limited, and for example an AAV1 vector, an AAV2 vector, an AAV6 vector, an AAV7 vector, an AAV8 vector, an AAV9 vector, an AAV10 vector, an AAV11 vector, or an AAV12 vector that are known to have tropism towards muscle tissue (skeletal muscle, cardiac muscle, and/or smooth muscle) may be employed.
  • Treatment of DM1 employing the present invention may be an aspect of directly applying the present invention to the living body of a subject suffering from DM1 (in vivo), or may be an aspect of treating the cells or tissues outside the living body with the present invention and then introducing into the living body of a subject (ex vivo).
  • the method for producing an expression vector comprising a nucleic acid encoding the PPR protein of the present invention is not limited, and production can be done by those skilled in the art by conventional means (such as use of commercially available protein expression vector production kits, use of commercially available viral expression vector production kits, and consignment to manufacturing companies).
  • the method for producing the PPR protein of the present invention is not limited, and for example, a desired PPR protein can be obtained by selectively extracting and/or purifying the PPR protein by conventional means from a cell (such as an animal cell, a plant cell, E. coli , and yeast) comprising an expression vector (such as a plasmid, a transposon, and a virus) encoding the PPR protein designed by the methods described herein.
  • a cell such as an animal cell, a plant cell, E. coli , and yeast
  • an expression vector such as a plasmid, a transposon, and a virus
  • CUG-PPR CUG repeat RNA sequence
  • a gene fused in the order of luciferase, PPR protein, and 3 ⁇ hexahistidine tag was cloned into multiple cloning sites of E. coli expression plasmid vector pET-22b.
  • the expression of the fusion gene is controlled by a T7 promoter.
  • Cloning of the gene having the correct size was confirmed by PCR method, and the gene sequence was confirmed by sequencing (SEQ ID NOs. 11-14). These were designated Luc-CUG-PPR 1, Luc-CUG-PPR 2, Luc-CUG-PPR 3, and Luc-CUG-PPR 4, and employed in subsequent tests.
  • the expression plasmid vector constructed as above was gene transferred into E. coli Rosetta (DE3) strain.
  • This E. coli was cultured in 2 mL of LB medium containing 100 ⁇ g/ ⁇ L ampicillin at 37° C. for 12 hours. The culture medium was transferred to an incubator at 15° C. when OD 600 reached 0.5 to 0.8, and left still for 30 minutes. Then, 100 ⁇ L (final concentration 0.1 mM IPTG) was added, and this was cultured at 15° C. for 16 hours. Centrifugation was performed at 5,000 ⁇ g, 4° C., for 10 minutes, the E.
  • coli pellets were collected, 1.5 mL of lysis buffer (20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40, 1 mM MgCl 2 , 2 mg/ml lysozyme, 1 mM PMSF, 2 ⁇ l of DNase) was added, and this was frozen at ⁇ 80° C. for 20 minutes. Frozen homogenization of cells was carried out at 25° C. for 30 minutes with shaking. Subsequently, centrifugation operation was performed at 3700 rpm, 4° C., for 15 minutes, and the supernatant comprising soluble PPR protein ( E. coli lysate) was collected and employed for the following experiment.
  • lysis buffer 20 mM Tris-HCl, pH 8.0, 150 mM NaCl, 0.5% NP-40, 1 mM MgCl 2 , 2 mg/ml lysozyme, 1 mM PMSF, 2 ⁇ l of DNase
  • RNA probes were synthesized to have biotin modified at the 5′-terminal of each of a 30-base RNA comprising the target CUG ⁇ 7 sequence
  • blocking buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM MgCl 2 , 0.5% NP-40, 1 mM DTT, 1% BSA) was added, and the plate surface was blocked for 30 minutes at room temperature.
  • One hundred microliters of the E. coli lysate comprising luciferase-fusion PPR protein having 1.5 ⁇ 10 8 LU/ ⁇ L of luminescence amount was added to the wells, and binding reaction was performed for 30 minutes at room temperature.
  • washing buffer (20 mM Tris-HCl (pH 7.6), 150 mM NaCl, 5 mM MgCl 2 , 0.5% NP-40, 1 mM DTT) was performed 5 times, 40 ⁇ L of the luciferase substrate (Promega, E151A) diluted 2500-folds with the washing buffer was added to the wells and allowed to react for 5 minutes, and then the amount of luminescence was measured with a plate reader (PerkinElmer, Cat No. 5103-35).
  • PPR protein was allowed to act on DM1 model cells to measure the number of RNA aggregates with FISH (fluorescent in situ hybridization) method.
  • C2C12-DMPK800R Nucleic Acids Research, 2014, 42(10), 6591-6602
  • C2C12-DMPK800 was produced with the following procedure.
  • a plasmid pLC16
  • pLC16 a plasmid comprising a sequence having 800 CTGs inserted into the 3′ untranslated region of the DMPK gene and a plasmid that expresses PhiC31 integrase were both transfected to mouse myoblast strain C2C12 with Nucleofector (product name, from Lonza), and then stable expression strains were selected with a selection medium supplemented with puromycin.
  • plasmid that expresses Cre recombinase was transfected to the stable expression strain, and clones in which transcription of mRNA having 800 CUG repeats is induced were selected with a selection medium supplemented with hygromycin.
  • a fusion gene in the order of green fluorescent protein mCLover3 gene, PPR protein gene, 3 ⁇ nucleus local signal, 3 ⁇ FLAG epitope tag was cloned into a mammal expression plasmid vector.
  • the expression of the fusion gene is controlled by a CMV promoter and a SV40 poly-A signal. Cloning of the gene having the correct size was confirmed by PCR method, and the gene sequence was confirmed by sequencing.
  • a fusion gene that does not comprise the PPR protein gene i.e., a fusion gene of green fluorescent protein mCLover3 gene, 3 ⁇ nucleus local signal, and 3 ⁇ FLAG epitope tag, was prepared with the same method.
  • PPR protein is underlined mCLo-CUG- MAGVSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICT PPR1 TGKLPVPWPTLVTTFGYGVACFSRYPDHMKQHDFFKSAMPEGYVQERTISF (SEQ ID NO. 17) KDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHYVYTT (SEQ ID NO.
  • DM1 model cells were cultured with DMEM medium containing 10% FBS and penicillin/streptomycin at 37° C. under 5% CO 2 condition.
  • DMEM medium containing 10% FBS and penicillin/streptomycin
  • Two hundred nanograms of the plasmid DNA constructed as above, 0.6 ⁇ L FugeneTM-HD (Promega, E2311), 200 ⁇ L Opti-MEM were mixed, the whole amount was added to the wells, cultured at 37° C. under 5% CO 2 environment for 72 hours, and then fixed with 3% paraformaldehyde for 15 minutes at room temperature. After fixation, this was washed twice with PBS, and then permeabilized with PBS containing 0.5% TritonX-100 for 5 minutes.
  • PPR protein was applied to DM1 model cells, and it was verified whether or not the splicing abnormality at exon 22 of the Atp2a1 gene improved.
  • Example 2 The same DM1 model cells as in Example 2 (C2C12-DMPK800R) was used.
  • Example 2 The same expression plasmid vector as in Example 2 was used.
  • PPR protein expression plasmid vector was gene transferred into DM1 model cells via the same method as in Example 2, and after 72 hours, total RNA was extracted with RNeasy Mini Plus Kit (product name, from Qiagen). As control, DM1 model cells without gene transfer of PPR protein expression plasmid vector were employed, and total RNA was similarly extracted. Subsequently, cDNA was prepared with SuperScript III First Strand Synthesis System (from Invitrogen) After subjecting the cDNA to RNaseH treatment, RT-PCR was performed with the following Atp2a1 exon 22 RT primers.
  • the RT-PCR product was subjected to electrophoresis with 2% agarose gel, stained with GelRed, and then normal type PCR products and abnormal type PCR products were each quantified with an image analyzer (ChemiDoc Touch Imaging System, from BioRad).
  • Example 2 The same DM1 model cells as in Example 2 (C2C12-DMPK800R) was used.
  • PPR protein expression plasmid vector was gene transferred into DM1 model cells via the same method as in Example 2.
  • the medium was exchanged to a differentiation medium, and 72 hours after transfection, nucleus staining by DAPI as well as antibody staining with anti-MyHC antibody were carried out.
  • Antibody staining was first carried out, cells having 2 or more nuclei inside the cell were further set as myotube cells, and the total number of nuclei inside the myotube cells confirmed in the observation field was counted (hereinafter referred to as value A).
  • the total number of nuclei in the whole field including not only the myotube cells in the above passage but also undifferentiated cells was counted (hereinafter referred to as value B).
  • the Fusion index represents the percentage of value A divided by value B, and means the muscle differentiation efficiency.
  • AAV6 loaded with the CUG-PPR gene was administered in single dose into the anterior tibial muscle, and the effect on myotonia symptoms in the anterior tibial muscle, the effect on the splicing abnormality of the Atp2a1 gene and the skeletal muscle-type chloride channel (Clcn1) gene, as well as the number of RNA aggregate formation were verified.
  • HSA LR mice (granted from Dr. Charles Thornton (University of Rochester) and bred in the laboratory animal facility at University of Osaka) were used.
  • HSA LR mouse is a transgenic animal having the CTG repeat sequence elongated 220 times at the 3′ untranslated region of the hACTA gene (human gene constitutively expressed in myocytes) integrated into the genome, and the symptoms of DM1 appear when the mRNA having the CUG sequence elongated is expressed in myocytes (Science, 2000, 289 (5485), 1769-73).
  • wild-type mice FVB/NJcl mice, purchased from CLEA Japan
  • AAV vector component of AAVproTM Helper Free System (AAV6), Takara, 6651
  • AAV6 component of AAVproTM Helper Free System
  • a gene having green fluorescent protein mCLover3, PPR protein, 3 ⁇ nuclear localization signal, and 3 ⁇ FLAG epitope tag fused in order was inserted. Verification of the expression size of the constructed gene by PCR, and verification of the inserted sequence by sequencing was performed. The expression of the fusion gene is controlled by the CMV promoter and the Human growth hormone polyA signal.
  • AAV vector constructed in the preceding section Twenty micrograms each of the AAV vector constructed in the preceding section, the pRC6 vector and the phelper vector (both components of AAVproTM Helper Free System (AAV6), Takara, 6651) were gene transferred into 2.5 ⁇ 10 8 cells of the AAVproTM HEK293T cell line (Takara, 632273) with Polyethylenimine, Linear, MW 25000 (Polysciences, Inc., 23966-1). Three days after transfection, AAV producer cells were obtained. AAVproTM Purification Kit Maxi (all serotypes) (Takara, 6666) was employed for AAV purification. Titration was carried out with AAVpro titration kit for real time PCR ver.
  • AAVproTM Purification Kit Maxi all serotypes
  • the dosage of AAV was 1 ⁇ 10 10 vg/leg and administered in single dose.
  • the anterior tibial muscle was collected from the mice and sections were produced. These were washed twice with PBS, and then permeabilized with PBS containing 0.5% TritonX-100 for 5 minutes. Subsequently, these were subjected to 10 minutes of prehybridization treatment with 2 ⁇ SSC buffer containing 30% formamide. Then, these were subjected to 1 hour of hybridization treatment at 37° C. with 2 ⁇ SSC buffer containing 30% formamide, 2 ⁇ g/mL BSA, 66 ⁇ g/mL yeast tRNA, 2 mM vanadyl complex (vanadyl complex), and 1 ng/ ⁇ L Texas Red CAG probe.
  • the RT-PCR product was subjected to electrophoresis with 2% agarose gel, stained with GelRed, and then normal type PCR products and abnormal type PCR products were each quantified with an image analyzer (ChemiDoc Touch Imaging System, from BioRad), and the proportion of the normal type in the RT-PCR product was calculated. T-test was employed for statistical analysis.
  • the analytical results of myotonia are shown in FIG. 7 .
  • myotonia of Severity 2 was detected in all five out of five mice.
  • AAV6-mCLo-CUG-PPR 1 administration DM1 group and the AAV6-mCLo-CUG-PPR 2 administration DM1 group two out of five mice showed Severity 1 and the remaining three mice showed Severity 2.
  • the improved two cases were from the experiment group with high improvement effect for Clcn1 splicing abnormality ( FIG. 8 ).
  • AAV9 loaded with the CUG-PPR gene was administered in single dose to DM1 model mice in the tail vein at each amounts of 3 ⁇ 10 13 , 1 ⁇ 10 14 , and 3 ⁇ 10 14 vg/kg, and the effect on myotonia symptoms in the femoral muscle, the effect on the splicing abnormality of the Atp2a1 gene and the skeletal muscle-type chloride channel (Clcn1) gene, as well as the number of RNA aggregate formation were verified.
  • HSA LR mice (granted from Dr. Charles Thornton (University of Rochester) and bred in the laboratory animal facility at University of Osaka) were used.
  • HSA LR mouse is a transgenic animal having the CTG repeat sequence elongated 220 times at the 3′ untranslated region of the hACTA gene (human gene constitutively expressed in myocytes) integrated into the genome, and the symptoms of DM1 appear when the mRNA having the CUG sequence elongated is expressed in myocytes (Science, 2000, 289 (5485), 1769-73).
  • wild-type mice FVB/NJcl mice, purchased from CLEA Japan
  • a gene having PPR protein and 3 ⁇ nuclear localization signal fused in order was inserted. Verification of the expression size of the constructed gene by PCR, and verification of the inserted sequence by sequencing was performed. The expression of the fusion gene is controlled by the CMV promoter and the Human growth hormone polyA signal.
  • the four groups of the AAV9-CUG-PPR 1 low dose administration group (hereinafter abbreviated as Low dose: LD), the AAV9-CUG-PPR 1 medium dose administration group (hereinafter abbreviated as Middle dose: MD), the AAV9-CUG-PPR 1 high dose administration group (hereinafter abbreviated as High dose: HD), and the PBS administration group (hereinafter abbreviated as PBS) were set.
  • n 5 for each group (3 male and 2 female mice).
  • the dosage of AAV was 3 ⁇ 10 13 vg/kg for LD, 1 ⁇ 10 14 vg/kg for MD, and 3 ⁇ 10 14 vg/kg for HD, and administered in single dose in the tail vein.
  • To the PBS administration group the same volume of PBS was administered in single dose.
  • RNA aggregates inside the nucleus was counted with a fluorescence microscope (Keyence PZ-9000).
  • the RT-PCR product was subjected to electrophoresis with 2% agarose gel, stained with GelRed, and then normal type PCR products and abnormal type PCR products were each quantified with an image analyzer (ChemiDoc Touch Imaging System, from BioRad), and the proportion of the normal type in the RT-PCR product was calculated. T-test was employed for statistical analysis.
  • the electrical myotonia phenomenon of the mouse femoral muscle was analyzed with needle electromyography under anesthesia. Specifically, the occurrence frequency of electrical myotonia phenomenon when a needle electrode was inserted 20 times into the mouse femoral muscle was evaluated in the following 4 stages.
  • the resected femoral muscle organ fragment was wet with 800 ⁇ L of the TRIzol Reagent. Two homogenization beads (TOMY, SUB-30) were added, and the organ was homogenized with a beads cell homogenizing device Micro Smash (TOMY SEIKO, MS-100R). Six hundred microliters of the supernatant obtained after centrifugation at 4° C., 15,000 g for 5 minutes were collected, 160 ⁇ L of chloroform solution was added, and this was mixed by inversion at room temperature for 5 minutes. Then, this was centrifugated at 4° C., 15,000 g for 5 minutes, and separated into the aqueous phase and the organic phase.
  • TOMY SEIKO beads cell homogenizing device Micro Smash
  • RNA Three hundred microliters of the aqueous phase were collected, and this was subjected to nucleic acid purification device Maxwell RSC Instrument to obtain a purified total-RNA product. The concentration and the purity of the RNA was confirmed by carrying out absorbance measurement with NanoDrop 8000. In order to verify the degree of degradation of the RNA obtained, 20-250 ng/ ⁇ L of the total-RNA was used to perform electrophoresis by LabChip GX Touch HT (PerkinElmer, CLS137031J), and it was confirmed from the band strength ratio of 28S and 18S rRNA that degradation had not proceeded.
  • RNA 20-100 ng/ ⁇ L of the RNA obtained was subjected to reverse transcription reaction according to the product protocol of SuperScript III Reverse Transcriptase (Thermo Fisher Scientific (Life Technologies), 18080085).
  • SuperScript III Reverse Transcriptase Thermo Fisher Scientific (Life Technologies), 18080085.
  • the cDNA produced, Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent, 600882), and the primers shown below were mixed according to manual, and then amplified with real-time PCR system Aria MX (Agilent) to perform quantitative analysis.
  • RNA-foci analysis The results of RNA-foci analysis are shown in FIG. 9 .
  • the femoral muscle organ sections obtained were employed to carry out RNA-FISH, and the RNA-foci formation suppression effect by AAV9-CUG-PPR 1 was verified.
  • dose-dependent suppression effect on the formation of RNA-foci was confirmed in the AAV9-CUG-PPR 1 administration group.
  • formation suppression effect observed were 39% for the PBS administration group, 18% for the AAV9-CUG-PPR 1 low dose group, 23% for the AAV9-CUG-PPR 1 medium dose group, and 17% for the AAV9-CUG-PPR 1 high dose administration group.
  • the evaluation results of splicing abnormality are shown in FIG. 10 .
  • Total RNA was collected from the organ obtained, reverse transcription PCR was performed, and abnormal splicing of the Clcn1 gene and the Atp2a1 gene were evaluated.
  • the abnormal splicing phenomenon of Clcn1 is the Cl channel which is known to be the direct cause of myotonic discharge phenomenon.
  • Atp2a1 is a Ca transporter likewise related to myotonic discharge, and is known to be a sensitive marker gene.
  • the PBS administration group showed 58% normal splicing, whereas the AAV9-CUG-PPR 1 low dose group showed 72%, the AAV9-CUG-PPR 1 medium dose group showed 78%, and the AAV9-CUG-PPR 1 high dose group showed 82%, confirming a dose-dependent splicing improvement.
  • the PBS administration group showed 25% normal splicing, whereas the AAV9-CUG-PPR 1 low dose group showed 56%, the AAV9-CUG-PPR 1 medium dose group showed 60%, and the AAV9-CUG-PPR 1 high dose group showed 64%, showing a dose-dependent splicing improvement.
  • the decrease improvement effect of myotonic discharge by PPR was represented by a score in 4 stages based on the number of myotonic discharge.
  • Score 0 means detection of myotonic discharge in 0 out of 20 times
  • Score 1 means 1-9 times out of 20
  • Score 2 means 10-19 times out of 20
  • Score 3 means 20 out of 20 times.
  • the PBS administration group showed Score 3
  • the AAV9-CUG-PPR 1 low dose group showed Score 3 in 1/5 case and Score 2 in 4/5 cases
  • the medium dose group showed Score 2 in 4/5 cases and Score 1 in 1/5 case
  • the low dose group showed improvement to Score 2 in 3/5 cases and Score 1 in 2/5 cases ( FIG. 11 ).
  • RNA was extracted from the analytical organ femoral muscle and quantitative PCR of PPR mRNA was carried out.
  • dose-dependent expression of PPR mRNA was confirmed ( FIG. 12 ).
  • the relative value when the value of the individual with the lowest signal in the AAV9-CUG-PPR 1 low dose administration group was set as 1 using GAPDH as the internal standard was used. Taking the median of each group, the PBS administration group was 0.2, the low dose administration group was 0.7, the medium dose administration group was 1.8, and the high dose administration group was 2.6, confirming a dose-dependent PPR expression ( FIG. 12 ).
  • AAV9 loaded with the CUG-PPR gene was administered in single dose to DM1 model mice in the tail vein, and the effect on myotonia symptoms in the femoral muscle at each time point, the effect on the splicing abnormality of the Atp2a1 gene and the skeletal muscle-type chloride channel (Clcn1) gene, as well as the number of RNA aggregate formation were verified.
  • HSA LR mice (granted from Dr. Charles Thornton (University of Rochester) and bred in the laboratory animal facility at University of Osaka) were used.
  • HSA LR mouse is a transgenic animal having the CTG repeat sequence elongated 220 times at the 3′ untranslated region of the hACTA gene (human gene constitutively expressed in myocytes) integrated into the genome, and the symptoms of DM1 appear when the mRNA having the CUG sequence elongated is expressed in myocytes (Science, 2000, 289 (5485), 1769-73).
  • wild-type mice FVB/NJcl mice, purchased from CLEA Japan
  • a gene having PPR protein and 3 ⁇ nuclear localization signal fused in order was inserted. Verification of the expression size of the constructed gene by PCR, and verification of the inserted sequence by sequencing was performed. The expression of the fusion gene is controlled by the CMV promoter and the Human growth hormone polyA signal.
  • the production of the AAV vector constructed in the preceding section was consigned to SignaGen Laboratories (hereinafter abbreviated as SG Company).
  • Packaging was carried out by co-transfecting the packaging cell strain with the Rep/Cap plasmid and the Helper plasmid from SG Company and the AAV vector sent from the inventors' company.
  • the cell homogenate comprising the packaged AAV was purified by density gradient centrifugation and titrated.
  • the AAV obtained was 1.16 ⁇ 10 14 vg/ml and the volume was 5 ml. This was employed in the subsequent experiment as AAV9-CUG-PPR 1.
  • the sequences of the PPR proteins (PPR 1) encoded in each AAV vector are identical to the sequences of the PPR proteins described in Table 1.
  • mice 14, 28, 56, or 112 days after the final administration, the femoral muscle was collected from the mice and sections were produced. These were washed twice with PBS, and then permeabilized with PBS containing 0.5% TritonX-100 for 5 minutes. Subsequently, these were subjected to 10 minutes of prehybridization treatment with 2 ⁇ SSC buffer containing 30% formamide. Then, these were subjected to 1 hour of hybridization treatment at 37° C. with 2 ⁇ SSC buffer containing 30% formamide, 2 ⁇ g/mL BSA, 66 ⁇ g/mL yeast tRNA, 2 mM vanadyl complex (vanadyl complex), and 1 ng/ ⁇ L Texas Red CAG probe.
  • RNA aggregates inside the nucleus was counted with a fluorescence microscope (Keyence PZ-9000).
  • the RT-PCR product was subjected to electrophoresis with 2% agarose gel, stained with GelRed, and then normal type PCR products and abnormal type PCR products were each quantified with an image analyzer (ChemiDoc Touch Imaging System, from BioRad), and the proportion of the normal type in the RT-PCR product was calculated. T-test was employed for statistical analysis.
  • the electrical myotonia phenomenon of the mouse femoral muscle was analyzed with needle electromyography under anesthesia. Specifically, the occurrence frequency of electrical myotonia phenomenon when a needle electrode was inserted 20 times into the mouse femoral muscle was evaluated in the following 4 stages.
  • RNA-foci analysis The results of RNA-foci analysis are shown in FIG. 13 .
  • the femoral muscle organ sections obtained were employed to carry out RNA-FISH, and the RNA-foci formation suppression effect by AAV9-CUG-PPR 1 was verified.
  • dose-dependent suppression effect on the formation of RNA-foci was confirmed in the AAV9-CUG-PPR 1 administration group.
  • the value of the PBS administration group employed in the dose-dependency test of Example 6 was used as control.
  • RNA-foci positive cell rate formation suppression effect confirmed were 39% for the control group, 36% for the Day 14 after administration of AAV9-CUG-PPR 1 group, 23% for the Day 28 after administration group, 19% for the Day 56 after administration group, and 18% for the Day 112 after administration group ( FIG. 13 ).
  • the evaluation results of splicing abnormality are shown in FIG. 14 .
  • Total RNA was collected from the organ obtained, reverse transcription PCR was performed, and abnormal splicing of the Clcn1 gene and the Atp2a1 gene were evaluated.
  • the abnormal splicing phenomenon of Clcn1 is the Cl channel which is known to be the direct cause of myotonic discharge phenomenon.
  • Atp2a1 is a Ca transporter likewise related to myotonic discharge, and is known to be a sensitive marker gene.
  • the PBS administration group shown in Example 6 showed 58% normal splicing, whereas the experiment group of Day 14 after administration of AAV9-CUG-PPR 1 showed 62%, Day 28 after administration of AAV9-CUG-PPR 1 showed 76%, and Day 56 after administration of AAV9-CUG-PPR 1 showed 79%, and 80% splicing improvement was confirmed for Day 112 after administration of AAV9-CUG-PPR 1.
  • the PBS administration group shown in Example 6 showed 25% normal splicing, whereas the experiment group of Day 14 after administration of AAV9-CUG-PPR 1 showed 30%, Day 28 after administration of AAV9-CUG-PPR 1 showed 63%, and Day 56 after administration of AAV9-CUG-PPR 1 showed 76%, and 75% splicing improvement was confirmed for Day 112 after administration of AAV9-CUG-PPR 1 ( FIG. 14 ).
  • the decrease improvement effect of myotonic discharge by PPR was represented by a score in 4 stages based on the number of myotonic discharge.
  • Score 0 means detection of myotonic discharge in 0 out of 20 times
  • Score 1 means 1-9 times out of 20
  • Score 2 means 10-19 times out of 20
  • Score 3 means 20 out of 20 times.
  • the PBS administration group shown in Example 6 showed Score 3
  • the experiment group of Day 14 after administration of AAV9-CUG-PPR 1 showed Score 3 in 4/5 cases and Score 2 in 1/5 case
  • the experiment group of Day 28 after administration of AAV9-CUG-PPR 1 showed Score 2 in 5/5 cases
  • the experiment group of Day 56 after administration of AAV9-CUG-PPR 1 showed Score 2 in 3/5 cases and Score 1 in 2/5 cases
  • the experiment group of Day 112 after administration showed Score 2 in 3/5 cases and Score 1 in 2/5 cases ( FIG. 15 ).
  • AAV9-CUG-PPR 1-administered mice showed improvement of drug effect until 14 days, 28 days, and 56 days after administration, and the drug effect still continued at Day 112 after administration.
  • Example 8 Comprehensive Improvement of DM1-Related Splicing Abnormality by AAV9-CUG-PPR 1 Administration
  • AAV9-CUG-PPR 1 was administered in single dose at 3 ⁇ 10 14 vg/kg in the tail vein, and the femoral muscle tissue fragment resected 8 weeks after administration (Day 56) was set as the PPR therapy group.
  • the same volume of PBS was administered in single dose, and the resected femoral muscle was set as the PPR untreated group.
  • the femoral muscle tissue fragment of a wild-type FVB/NJcl mouse having similar genetic background was employed as the wild-type group.
  • RNA Sequence Illumina Hiseq 2 ⁇ 150 bp sequence.
  • ribosomal RNA in the total RNA was removed by Poly A selection method.
  • GRCm39 obtained from Ensemble
  • alignment was performed on the lead sequence after trimming with STAR (v2.7.9a).
  • Specification of splicing events was detected with rMATS (v3.2.5) based on the STAR-aligned file (.bam).
  • rMATS v3.2.5
  • STAR-aligned file .bam
  • comparison of the wild-type group and the PPR untreated group HALR mice administered with PBS was performed, the presence or absence of variation was judged with “p-adjusted-value” and “InclLevelDifference” as indicators, and only the candidates that were defined to be varied were extracted.
  • the Percent spliced-in index was defined as the proportion of the amount of spliced-in transcript against the total lead number of the target gene. Metascape (https://metascape.org/gp/index.html #/main/step1) was used for gene ontology analysis.
  • RNA was extracted from the femoral muscle resected from wild-type mice (14 weeks-old) described in the preceding section (hereinafter referred to as the wild-type group), the femoral muscle resected 8 weeks after PBS administration to HSALR mice (hereinafter referred to as the PPR untreated group), and further the femoral muscle resected 8 weeks (Day 56) after AAV9-CUG-PPR 1 administration (hereinafter the PPR treatment group), and exhaustive analysis of expressed RNA was carried out by next-generation sequencing technology.
  • PSI percent spliced-in index
  • Gene ontology analysis was performed on the gene group with abnormal splicing, and they were the terms related to muscle function or muscle differentiation. As a result of analyzing the number of genes in each gene ontology that achieved recognition of improvement, it was found that genes achieving recognition of strong improvement (greater than 50%) was about 40%, coming to a total of 90% including those achieving recognition of improvement (greater than 20%) ( FIG. 16 c ).
  • organ fragments (heart, femoral muscle, gastrocnemius muscle, and anterior tibial muscle) resected from normal mice iv-administered with AAV9-CUG-PPR 1 (administration dosage: 1 ⁇ 10 13 vg/kg, 3 ⁇ 10 13 vg/kg, or 3 ⁇ 10 14 vg/kg) were used.
  • Administration dosages were 1 ⁇ 10 13 vg/kg, 3 ⁇ 10 13 vg/kg, or 3 ⁇ 10 14 vg/kg and administration was performed in the tail vein, and the volume was set to be 10 ⁇ L/g. Observation duration was set to be 28 days after administration.
  • the heart, the femoral muscle, the gastrocnemius muscle, and the anterior tibial muscle was resected from each mouse individual to perform analysis of PPR mRNA.
  • 50 mg ( ⁇ 5 mg) fragments of the organ fragments obtained in the preceding section were wet with 800 ⁇ L of the TRIzol Reagent.
  • Two homogenization beads (TOMY, SUB-30) were added, and the organs were homogenized with a beads cell homogenizing device Micro Smash (TOMY SEIKO, MS-100R), and this was centrifugated at 4° C., 15,000 g for 5 minutes.
  • TOMY SEIKO beads cell homogenizing device Micro Smash
  • RNA 20-100 ng/ ⁇ L of the RNA obtained was subjected to reverse transcription reaction according to the product protocol of SuperScript III Reverse Transcriptase (Thermo Fisher Scientific (Life Technologies), 18080085).
  • Quantitative PCR was carried out with real-time PCR system Aria MX (Agilent) according to the general manual of Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent, 600882).
  • the results of quantitative PCR are shown in FIG. 17 .
  • a rise in dose-dependent PPR expression amount in each tissue was confirmed, and a maximum expression amount was shown in the maximally administered 3 ⁇ 10 14 vg/kg administration group.
  • AAV9-CUG-PPR 1 can be systematically delivered and expressed, and suggests that it is effective for DM1 which is a multi-organ disease that affects the heart in addition to skeletal muscles and smooth muscles.
  • Example 10 Analysis of PPR mRNA Expression Persistency in Each Organ of Normal Mice that were Applied AAV9-CUG-PPR 1
  • organ fragments cortisol, femoral muscle, gastrocnemius muscle, and anterior tibial muscle resected from normal mice iv-administered with AAV9-CUG-PPR 1 (administration dosage: 1 ⁇ 10 14 vg/kg) were used.
  • Administration dosage was 1 ⁇ 10 14 vg/kg and administration was performed in the tail vein, and the volume was set to be 10 ⁇ L/g.
  • Observation duration was set to be Day 14 after administration, Day 28 after administration, Day 56 after administration, Day 112 after administration, and Day 182 after administration.
  • the heart, the femoral muscle, the gastrocnemius muscle, and the anterior tibial muscle was resected from each mouse individual to perform analysis of PPR mRNA.
  • 50 mg ( ⁇ 5 mg) fragments of the organ fragments obtained in the preceding section were wet with 800 ⁇ L of the TRIzol Reagent.
  • Two homogenization beads (TOMY, SUB-30) were added, and the organs were homogenized with a beads cell homogenizing device Micro Smash (TOMY SEIKO, MS-100R).
  • TOMY SEIKO beads cell homogenizing device Micro Smash
  • RNA obtained was subjected to reverse transcription reaction according to the product protocol of SuperScript III Reverse Transcriptase (Thermo Fisher Scientific (Life Technologies), 18080085). Quantitative PCR was carried out with real-time PCR system Aria MX (Agilent) according to the general manual of Brilliant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent, 600882).
  • the present invention may exert the desired effect for a long time with fewer number of administrations to the subject.

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Jauvin, et al. (2017) "Targeting DMPK with Antisense Oligonucleotide Improves Muscle Strength in Myotonic Dystrophy Type 1 Mice", Molecular Therapy: Nucleic Acids, 7: 465-74. (Year: 2017) *
Tang, et al. (2010) "AAV-directed muscular dystrophy gene therapy", Expert Opinion in Biological Therapy, 10(3): 395-408. (Year: 2010) *
Zhang, et al. (2014) "Treatment of Type 1 Myotonic Dystrophy by Engineering Site-Specific RNA Endonucleases that Target (cug)n Repeats", Molecular Therapy, 22(2): 312-20. (Year: 2014) *

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